Nanovolume microcapillary crystallization system

ABSTRACT

A nanovolume microcapillary crystallization system allows nanoliter-volume screening of crystallization conditions in a crystal card that allows crystals to either be removed for traditional cryoprotection or in situ X-ray diffraction studies on protein crystals that grow within. The system integrates formulation of crystallization cocktails with preparation of the crystallization experiments. The system allows the researcher to select either gradient screening in crystallization experiments for efficient exploration of crystallization phase space or a combination of sparse matrix with gradient screening to execute one comprehensive hybrid crystallization trial.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/484,983,filed Jun. 15, 2009, which claims the benefit of U.S. Provisional PatentApplication No. 61/061,536, filed Jun. 13, 2008, the entire disclosuresof which are incorporated herein by reference.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This subject matter was made, at least in part, with Government supportas provided for by the terms of NIGMS U54 GM074961, awarded by theNational Institute of General Medical Sciences. The Government hascertain rights in the subject matter.

BACKGROUND

The field of structural biology is generating technologies that increasethroughput and efficiency each year. Such advances have inspiredprogression from gene to three-dimensional structure in three days. Inan effort to improve efficiency, it is desirable to minimize the volumeof protein required such that sufficient material for crystallizationscreening and optimization can be obtained from cell-free synthesis.With the “three day” structure goal in mind, it is desirable to developseveral technologies to increase efficiency in the gene to structurepipeline.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

One aspect of the discussed subject matter includes a system for proteincrystallization, which comprises a pumping system and pieces of softwareconfigured to execute on the protein crystallization system to controlthe pumping system. The system further includes one or more crystalcards coupled to the pumping system, each configured to house a mixerand a microfluidic capillary that is coupled to the mixer to facilitatestorage and inspection of protein crystallization.

Another aspect of the subject matter includes a method for gradientscreening, which comprises regulating aqueous streams by independentlycontrolling each aqueous stream with a pumping system exercised bypieces of software. The method further comprises mapping outcrystallization phase space of a protein to illustrate transition fromprecipitation, to microcrystals, to single crystals in a proteincrystallization experiment.

A further aspect of the subject matter includes a method for hybridscreening, which comprises pre-forming precipitant plugs and pre-formingplug spacers, each separating two precipitant plugs from each other. Themethod further comprises forming gradients by merging precipitant plugs,plug spacers, and a protein stream. The method further includes mappingout crystallization phase space of a protein to illustrate transitionfrom precipitation, to microcrystals, to single crystals in a proteincrystallization experiment.

A further aspect of the subject matter includes a method which comprisesreceiving a crystal card with capillaries, coating capillaries with areagent to reduce the surface energy, and removing the reagent.

In another aspect, the subject matter includes a crystal card, whichcomprises a substrate configured to house a mixer circuit and aninspection circuit. The crystal card further includes a layer bonded tothe substrate and configured to peel from the substrate.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a block diagram illustrating an exemplary nanovolumemicrocapillary crystallization system;

FIG. 2 is a block diagram illustrating an exemplary pumping system ofthe nanovolume microcapillary crystallization system;

FIG. 3A is a pictorial diagram illustrating an exemplary user interfacefor configuring the pumping system;

FIG. 3B is a pictorial diagram illustrating an exemplary user interfacefor priming fluids to a crystal card of the system;

FIG. 3C is a pictorial diagram illustrating an exemplary user interfacefor specifying the production of nanoplugs in the crystal card whereinthe nanoplugs are of equal size and equal content according to oneembodiment of the subject matter;

FIG. 3D is a pictorial diagram illustrating an exemplary user interfacefor specifying the production of nanoplugs in the crystal card whereinthe nanoplugs have varying concentrations of protein and precipitantaccording to one embodiment of the subject matter;

FIG. 3E is a pictorial diagram illustrating an exemplary user interfacefor specifying the production of nanoplugs in the crystal card whereinthe nanoplugs have varying size and concentration for multipleprecipitants according to another embodiment of the subject matter;

FIG. 4A is a pictorial diagram illustrating a top isometric view of oneembodiment of a crystal card; FIG. 4B is a pictorial diagramillustrating a bottom isometric view of one embodiment of a crystalcard; FIG. 4C is a pictorial diagram illustrating a top view of oneembodiment of a crystal card; FIG. 4D is a pictorial diagramillustrating a side view of one embodiment of a crystal card; FIG. 4E isa pictorial diagram illustrating a bottom view of one embodiment of acrystal card;

FIG. 5A is a pictorial diagram illustrating a top isometric view ofanother embodiment of a crystal card; FIG. 5B is a pictorial diagramillustrating a bottom isometric view of another embodiment of a crystalcard; FIG. 5C is a pictorial diagram illustrating a top view of anotherembodiment of a crystal card; FIG. 5D is a pictorial diagramillustrating a side view of another embodiment of a crystal card; FIG.5E is a pictorial diagram illustrating a bottom view of anotherembodiment of a crystal card;

FIG. 6A is a pictorial diagram illustrating a top isometric view of athird embodiment of a crystal card; FIG. 6B is a pictorial diagramillustrating a bottom isometric view of a third embodiment of a crystalcard; FIG. 6C is a pictorial diagram illustrating a top view of a thirdembodiment of a crystal card; FIG. 6D is a pictorial diagramillustrating a side view of a third embodiment of a crystal card; FIG.6E is a pictorial diagram illustrating a bottom view of a thirdembodiment of a crystal card;

FIG. 7A is a pictorial diagram illustrating a top isometric view of afourth embodiment of a crystal card; FIG. 7B is a pictorial diagramillustrating a bottom isometric view of a fourth embodiment of a crystalcard; FIG. 7C is a pictorial diagram illustrating a top view of a fourthembodiment of a crystal card; FIG. 7D is a pictorial diagramillustrating a side view of a fourth embodiment of a crystal card; FIG.7E is a pictorial diagram illustrating a bottom view of a fourthembodiment of a crystal card;

FIG. 8A is a pictorial diagram illustrating a top isometric view of afifth embodiment of a crystal card; FIG. 8B is a pictorial diagramillustrating a bottom isometric view of a fifth embodiment of a crystalcard; FIG. 8C is a pictorial diagram illustrating a top view of anembodiment of a crystal card; FIG. 8D is a pictorial diagramillustrating a side view of a fifth embodiment of a crystal card; FIG.8E is a pictorial diagram illustrating a bottom view of a fifthembodiment of a crystal card;

FIG. 9A is a pictorial diagram illustrating a top isometric view of asixth embodiment of a crystal card; FIG. 9B is a pictorial diagramillustrating a bottom isometric view of a sixth embodiment of a crystalcard; FIG. 9C is a pictorial diagram illustrating an exploded isometricview of a sixth embodiment of a crystal card; FIG. 9D is a pictorialdiagram illustrating a top view of a sixth embodiment of a crystal card;FIG. 9E is a pictorial diagram illustrating a side view of a sixthembodiment of a crystal card; FIG. 9F is a pictorial diagramillustrating a bottom view of a sixth embodiment of a crystal card;

FIG. 10 is a pictorial diagram illustrating one embodiment of athree-plus-one mixer of one embodiment of a crystal card;

FIG. 11 is a pictorial diagram illustrating another embodiment of athree-plus-one mixer of one embodiment of a crystal card;

FIG. 12 is a pictorial diagram illustrating a third embodiment of athree-plus-one mixer of one embodiment of a crystal card;

FIG. 13 is a pictorial diagram illustrating a fourth embodiment of athree-plus-one mixer of one embodiment of a crystal card;

FIG. 14 is a pictorial diagram illustrating a cross section through oneembodiment of a crystal card; and

FIGS. 15A-15V are process diagrams illustrating an exemplary method forcrystallizing molecules using a nanovolume microcapillarycrystallization system.

DETAILED DESCRIPTION

Various embodiments of the subject matter describe a nanovolumemicrocapillary crystallization system which comprises a pump, softwareconfigured to control the pump, and a crystal card that houses a mixercircuit and an inspection circuit. The crystal cards are suitablymanufactured using materials that include one or more propertiesselected from a group consisting of X-ray transmission, optical clarity,moldability, chemical resistance and surface energy. The crystal cardshouse macromolecular crystals in various phases enabling eitherextraction of crystals from the crystal card or in situ X-raydiffraction. The crystals are promoted inside the crystal cards byformation of nanoplugs by the nanovolume microcapillary crystallizationsystem. Nanoplugs are formed by combining streams of aqueous solutionswith an immiscible and biologically inert carrier fluid, such asfluorocarbon solution. Streams of aqueous solutions, such as thosecomposed of a target molecule, buffer, and precipitant solutions, arecombined at the mixer circuit to form nanoplug crystallizationexperiments. The nanoplugs are incubated and monitored forcrystallization. Nanoplug crystallization experiments can be suitablyused to shed light on scientific questions regarding protein crystalnucleation and growth and to generate crystals for novel structuresolution.

The nanovolume microcapillary crystallization system facilitates twoscreening styles: gradient mode and hybrid mode. As used herein, theterm gradient mode includes any suitable screening method that providesvarious crystallization phases of molecules. The gradient mode allows acrystallographer to finely scan a crystal card to reveal crystallizationphase space of a particular molecule. Because each stream of aqueoussolution used in the nanovolume microcapillary crystallization systemcan be independently controlled using the pump via the software,concentration gradients of desired granularity over a series ofnanoplugs are suitably formed by changing the flow rates of theindividual streams. As a precipitant stream decreases in flow rate, thenanovolume microcapillary crystallization system increases a flow rateof a buffer stream such that that the sum of the flow rates remainsconstant. Using the gradient mode, crystallization phase space of aparticular molecule, such as a protein, can be mapped out to show atransition from precipitation, to microcrystals, to single crystals.

As an enhancement to gradient mode, hybrid mode combines gradients withsparse matrix screening on one crystal card. Sparse matrix screening ofmolecule crystals in nanoplugs can be achieved by generating apre-formed cartridge of different crystallizing agents. As used herein,the term hybrid mode includes hybrid screening, including any suitablescreening method that includes pre-formed cartridges. The hybrid modeextends the concept of sparse matrix screening by pre-formingprecipitant nanoplugs, separated by a nanoplug spacer (gas bubble), andforming a concentration gradient as they are merged with a moleculestream. Similar to the gradient mode, the hybrid mode generatesgradients by coordinating flow rate change between the pre-formedprecipitant nanoplugs and the buffer stream. By performing sparse matrixand gradient screening together on one crystal card, the hybrid mode isable to sample a large area of the crystallization phase space,generating 20-40 experiments from each pre-formed precipitant nanoplug.

As used hereinabove and hereinbelow, the term “nanoplug” refers to ananoliter-volume sized drop, such as a 10-20 nL aqueous drop, that fillsa microfluidic channel of the system. Each nanoplug comprises a distinctmicrocrystallization experiment. As used hereinabove and hereinbelow,the term “mixer circuit” means the inclusion of a circuit having threeaqueous channels and one carrier fluid channel that come together at apoint upstream of the inspection circuit of a crystal card. Additionalconfigurations are possible, such as mixers having four or five aqueouschannels and one carrier fluid channel. The aqueous channels cometogether and suitably intersect the carrier fluid channel at a 90 degreeangle. As used herein, the term “macro-micro interface” means theinclusion of a coupling between the syringes and a crystal card. In someembodiments, the syringes are connected to the mixer circuit or theinspection circuit via tubing, such as Teflon® (PTFE) tubing. In otherembodiments, the tubing is connected to the mixer circuit or theinspection circuit using connectors configured to fluidically connectthe inlets and outlets of the circuits with the tubing. As usedhereinabove and hereinbelow, the term “inspection circuit” refers to acapillary or channel where fluids come together and form aqueousnanoplugs that flow inside the capillary. The inspection circuit canalso be used to inspect the nanoplugs for crystal formation. Further,the inspection circuit can also be used to store crystals formed by themethods of the subject matter, and is therefore also referred to hereinas a storage capillary. As used herein, the term “main channel” refersto the area of the inspection circuit that locates downstream of themixer where the aqueous solutions and carrier fluid combine to formaqueous nanoplugs. As used hereinabove and hereinbelow, the term“molecule” includes small molecules, such as organic compounds and/orchemicals, and macromolecules. The term “biological molecule” refers toa molecule that is derived from, modeled on, or corresponds to amolecule from a biological source. The term also includes moleculessynthesized or produced in vitro, such as by cell-free synthesis, and/orin vivo, such as recombinant proteins, mutant proteins, and artificialproteins, natural and artificial nucleic acid molecules, and otherbiological molecules that do not occur in nature. As used herein, theterm “macromolecule” includes biopolymers such as nucleic acids,proteins, carbohydrates and lipids. For simplicity, the terms “protein”and “protein solution” are used herein to encompass other types ofmolecules in addition to proteins.

A nanovolume microcapillary crystallization system 100 useful formolecule crystallization is shown in FIG. 1. A prepared sample 102comprising a molecule for which the crystal structure is desired, forexample a prepared protein sample, is provided in an aqueous solution104. Additional aqueous solutions are also provided; for example, abuffer solution and a precipitant solution. The buffer solution maycomprise the buffer used to prepare the biological sample 102. A carrierfluid is also provided. The carrier fluid is immiscible with the aqueoussolutions. Suitable examples of carrier fluids include fluorinated oils;for example, FC-40 (3M Corp., St. Paul, Minn.). The aqueous solutionsand carrier fluid 104 are provided to one or more syringes 106 which areconnected to one or more pumps 108. The pump 108 is controlled bysoftware executed on a nanoplug-forming computer 110. The softwareexecuted on the nanoplug-forming computer 110 regulates the flow of theaqueous solutions and carrier fluid 104 in a crystal card 112. The flowof the aqueous solutions and carrier fluid in the crystal card 112 areobserved through a magnifying device such as a microscope 114.

A pumping system 200 useful for regulating the flow of various fluidsthrough a crystal card is shown in FIG. 2. Pump 1 202 controls syringe 1204 and syringe 2 208. Syringe 1 204 is loaded with an aqueous solutionsuch as buffer 206. Syringe 2 is filled with an aqueous solution such asa precipitant reagent 210. Pump 2 212 controls syringe 3 214 and syringe4 218. Syringe 3 214 is filled with an immiscible fluid such as acarrier fluid 216. Syringe 4 218 is filled with an aqueous solutioncontaining a molecule, such as a protein of interest 220. Suitable pumpsinclude Harvard Twin 33 syringe pumps (Harvard Apparatus, Holliston,Mass.). In some embodiments, the syringe pumps have been modified by themanufacturer to provide better accuracy. Suitable syringes includeHamilton syringes, such as an 1800 series Hamilton Gas Tight syringe.Suitable syringe volumes range from 10 ul to 100 ul. The pumping system200 is controlled by software executed on a nanoplug-forming computer110.

Suitable software is provided for controlling the pumping system 108.FIGS. 3A-3E illustrate representative user interfaces of the software ofthe system showing various modes that control the pumping system 200.FIG. 3A shows a representative user interface 300 of a configurationmode of the software. FIG. 3B shows a representative user interface 302of a prime mode of the software. FIG. 3C shows a representative userinterface 304 of a constant mode of the software. FIG. 3D shows arepresentative user interface 306 of a gradient mode of the software.FIG. 3E shows a representative user interface 308 of a hybrid mode ofthe software of the system.

Referring now to the crystal cards of the subject matter disclosedherein, a representative example of one embodiment of a crystal card isshown in FIGS. 4A-4E. The crystal card 400 is configured to be about thesame size as a standard microscope slide, being about 76.20 mm long andabout 25.40 mm wide (or about 3 inches long by about 1 inch wide). Thecrystal card 400 is about 1.0 to 1.5 mm thick. The crystal card ismanufactured of transparent polycarbonate by injection molding (SiloamBiosciences, Inc.).

Referring now to the embodiment shown in FIGS. 4A and 4B, the crystalcard 400 has an upper surface 402 and a lower surface 414 that isparallel to the upper surface 402. The crystal card 400 furthercomprises a substrate configured to house a mixer circuit 404 and astorage and inspection circuit 406. The mixer circuit 404 is comprisedof four microfluidic channels 421, 422, 424, and 426. See FIG. 4C.Channels 421, 422, and 424 come together and intersect channel 426 at a90 degree angle. Each channel comprises an inlet 410. See FIG. 4E. Theinspection circuit 406 comprises a long microfluidic capillary channelthat locates just downstream of the mixer 404 and ends at an outlet 412.The length of the microfluidic capillary 406 is about 67 cm. Themicrofluidic capillary channel 406 is also referred to as an inspectioncircuit, in which crystals produced in the card may be stored in thechannel 406 until subjected to in situ X-ray diffraction analysis orextracted for cryocooling. The microfluidic channels 421, 422, 424, 426and the capillary channel 406 are substantially square in cross-sectionand have an inner diameter of about 200 micrometers (μm)×200 μm.However, other configurations of the channels are possible.

Referring now to FIG. 4D, the crystal card 400 further comprises a layer420 that is thermally bonded to the substrate and configured to peelfrom the substrate. The peelable layer 420 is thermally bonded to thesubstrate surface 414. In other embodiments, the peelable layer 420 maybe chemically bonded to the substrate. The peelable layer 420 is about0.10 to 0.14 mm thick. The peelable layer 420 is suitably configuredsuch that removal of the peelable layer 420 exposes the interior spaceof the inspection circuit channel 406. The crystal card 400 furthercomprises a macro-micro interface that connects the syringes to thecrystal card. In one embodiment, the macro-micro interface includessections of rigid plastic tubing 430 (for example, tubing made of PEEK™polymer) that are connected at one end to the inlets 410 and outlet 412,and are connected at the other end to slip fit connectors 432 made offlexible silicone tubing. The slip fit connectors 432 are configured toaccept Teflon® tubing (PTFE) (not shown). The other end of the tubing isconnected to a syringe of the system. The Teflon® tubing has an innerdiameter of 360 um and an outer diameter of 760 um (ID/OD 360/760),whereas the connecter 432 has an inner diameter of 760 um, therebyforming a gas and liquid tight seal when the Teflon® tubing is insertedinto the connecter 432.

In operation, the channel 421 is connected to tubing that is filled withan aqueous solution, such as a buffer that is used in the proteinsolution of interest. Channel 422 is connected to tubing that is filledwith a precipitant solution. As used herein, it is understood that theterm precipitant is interchangeable with the term crystallant. Channel424 is connected to tubing that is filled with a solution containing atarget molecule of interest. In one embodiment, the target biologicalmolecule is a protein. Channel 426 is connected to tubing that is filledwith a carrier fluid. Suitable examples of carrier fluids includefluorinated oils or fluorocarbons, such as FC-40, although others arepossible. The carrier fluid is immiscible with the aqueous fluids andpreferentially wets the walls of the inspection circuit microchannel,thereby separating segments of the combined aqueous solution intonanoplugs that span the width of the channel. In one embodiment, theaqueous nanoplugs are about 10-20 nL in volume.

Referring now to FIGS. 5A-5E, a representative example of anotherembodiment of a crystal card of the subject matter is shown. Similarelements between different figures have similar reference numbers,wherein the first digit increases by one and corresponds to the figurenumber. For the sake of brevity, elements that are similar between thedifferent Figures will not be described further. In the embodiment shownin FIGS. 5A-5E, the inlet 510 is located in a shallow cylindricaldepression 508 located in a top surface 502 of the crystal card 500. Thecylindrical depression 508 is configured for attaching a connector (notshown) that connects tubing to the inlets 510 and outlet 512. Thedimensions of the crystal card 500 are shown in FIG. 5E. The crystalcard 500 is 76.2 mm long and 25.4 mm wide. The inlets 510 are spaced 4.5mm apart. The parallel channels of the inspection circuit 506 are 2.0 mmapart. However, as will be appreciated by a person skilled in the art,other suitable configurations are possible.

Referring now to FIGS. 6A-6E, a representative example of a thirdembodiment of a crystal card is shown. For the sake of brevity, similarelements that are described in previous figures are not described here.In the embodiment shown in FIGS. 6A-6E, the inlets 610 and outlet 612are positioned below a cylindrical projection 608 that is connected toand extends outwardly from the surface 602. The projection 608 isconfigured for attaching a connector (not shown) that connects tubing tothe inlets 610 and outlet 612. Digressing, the crystal cards illustratedin the embodiments shown in FIGS. 4-6 are manufactured from transparentpolycarbonate plastic by injection molding (Siloam Biosciences, Inc.).

Returning now to FIGS. 7-9, representative embodiments of a second typeof crystal card will be described. FIGS. 7A-7E illustrate arepresentative example of another embodiment of a crystal card of thesubject matter. For the sake of brevity, similar elements that aredescribed in previous figures are not described here. In the embodimentshown in FIGS. 7A-7E, the top surface 702 of the crystal card 700further comprises two rows of ports 708. The ports are configured toreceive a plastic connector (not shown) that is suitable for connectingtubing to the inlets 710 and outlets 712 located beneath the port 708.The surface 702 comprises 28 ports 708. However, different numbers ofports are possible depending on the design of the crystal card 700. Theport 708 extends about 2.5 mm above the surface 702 of the crystal card700. A hole is suitably drilled in the bottom center portion of the port708 such that it aligns with and is in fluidic connection with theinlets 710 and outlets 712. The center of the ports are spaced about 4.5mm apart. The hole drilled in the bottom of the port 708 is about 0.2 mm(200 um) in diameter. It will be understood that not every port isconnected to the circuit channels such that only desired ports toconnect tubing to the inlets 710 and the outlets 712 need be drilled. Inother embodiments, a laser is used to drill holes through the peelablelayer 720 before it is bonded to the bottom surface 714. Thelaser-drilled holes are configured to be in fluidic connection with theinlets 710 and the outlets 712. Tubing is connected to the laser-drilledholes using a specially designed crystal card holder (not shown).

Referring still to FIGS. 7A-7E, the crystal card further comprises twoseparate asymmetrical microfluidic channel circuits 706A, 706B. In 706A,the inspection circuit is about 270 mm long. In 706B, the inspectioncircuit is about 306 mm long. In both circuits 706A and 706, the outlet712 is located on the opposite side of the circuit from the inlets 710and the mixer circuits 704A, 704B. The embodiment shown in FIGS. 7A-7Ecomprises two separate configurations of the mixer circuit 704A, 704B.As shown in more detail in FIG. 10, the mixer circuit 704A comprises ashort neck region approximately 0.20 mm long between the aqueouschannels and the carrier fluid channel. As shown in more detail in FIG.11, the mixer circuit 704B lacks a neck region between the aqueouschannels and the carrier fluid channel. The mixer circuit 704A was foundto be suitable for aqueous nanoplug formation in a crystal card.

Referring now to FIGS. 8A-8E, a representative example of anotherembodiment of a crystal card of the subject matter is shown. For thesake of brevity, elements that are similar to previously describedelements are not further described here. The crystal card 800 comprisestwo separate symmetrical microfluidic channel circuits 806. In thisembodiment, the outlet 812 is located on the same side of the circuit806 as the mixer 804 and the inlets 810.

Referring now to FIGS. 9A-9E, a representative example of anotherembodiment of a crystal card of the subject matter is shown. For thesake of brevity, elements that are similar to previously describedelements are not further described here. A crystal card 900 comprises asingle microfluidic circuit comprising one mixer circuit 904 and a longinspection circuit 906. The inspection circuit 906 is about 665 mm long.FIG. 9C illustrates an exploded view of the crystal card 900. Piece 930comprising ports 908 is bonded to piece 940 comprising the microfluidiccircuit channels. The peelable layer 920 is thermally bonded to thebottom surface 914 of piece 940. However, in other embodiments, thepeelable layer 920 may be chemically bonded to the substrate surface914. The peelable layer 920 is suitably configured such that removal ofthe peelable layer 920 exposes the interior space of the inspectioncircuit channel 906. Digressing, the crystal cards illustrated in theembodiments shown in FIGS. 7-9 are manufactured from transparent cyclicolefin copolymer (COC) or comparable plastic (ThinXXS MicrotechnologyAG, Germany).

Returning now to FIGS. 10-13, representative examples of mixer circuitswill now be described. FIG. 10 shows a representative example of oneembodiment of a mixer circuit that corresponds to the mixer 704A shownin FIG. 7. The mixer circuit 1000 comprises three aqueous channels 1021,1022 and 1024. The aqueous channels are separated from the carrier fluidchannel 1026 by a neck region 1007. The channels are oriented such thatthe three channels 1021, 1022, 1024 containing aqueous solutions cometogether and intersect the channel 1026 containing the carrier fluid ata 90 degree angle. The mixer 1000 further comprises a portion of aninspection circuit 1006. Referring still to FIG. 10, the dimensions ofthe mixer 1000 will now be described. The neck region 1007 is about 0.2mm long. Channel 1021 is about 0.2 mm in diameter. Channels 1022, 1024are about 0.141 mm in diameter. Channels 1006, 1026 are about 0.2 mm indiameter. However, other suitable dimensions for a mixer circuit arepossible.

FIG. 11 shows a representative example of another embodiment of a mixercircuit that corresponds to the mixer circuit 704B shown in FIG. 7. Themixer circuit 1100 comprises three aqueous channels 1121, 1122 and 1124.The aqueous solution channels connect directly to the carrier fluidchannel 1106 in the absence of a neck region. The mixer circuit feedsinto the inspection circuit 1126. The channels are oriented such thatthe three channels containing aqueous solutions come together andintersect the channel containing the carrier fluid at a 90 degree angle.The diameter of channel 1121 is about 0.2 mm. The diameter of channels1122, 1124 is about 0.141 mm. The diameter of the junction regionbetween the aqueous channels and the carrier fluid channel 1126 is about0.285 mm. However, other suitable dimensions for a mixer are possible.

Referring now to FIG. 12, another view of the mixer circuit 704Adescribed in FIG. 7 is shown. The mixer circuit 1200 comprises threeaqueous channels 1221, 1222 and 1224 that are connected by a short neckregion to the carrier fluid channel 1226. The channels are oriented suchthat the three channels containing aqueous solutions come together andintersect the channel containing the carrier fluid at a 90 degree angle.Each channel has an inlet 1210. Downstream of the mixer circuit 1204,the solutions feed into a portion of an inspection circuit 1206.Referring still to FIG. 12, the dimensions of the mixer circuit 1200will now be described. The inlets 1210 are located about 4.4 mm from thechannels 1206, 1226. The aqueous channels 1221, 1222, 1224 make a rightangle turn about 2.9 mm from the inlet. The right angle turn has aninner radius R0.300 and an outer radius R0.500. The portion of channels1221, 1222, 1224 that are disposed in a plane parallel to channel 1206are about 1.300 mm from channel 1206. The aqueous channels 1221, 1222,1224 make a 45 degree turn before connecting with each other upstream ofthe neck region. The inner diameter of channel 1206 is about 0.200 mm(200 um). The parallel portions of channel 1206 are about 1.2 mm apart.However, other suitable dimensions are possible.

FIG. 13 shows a representative example of another embodiment of a mixerthat corresponds to the mixer circuits 804 and 904 shown in FIGS. 8 and9. The mixer circuit 1300 comprises aqueous channels 1321, 1322, and1324. The aqueous channels are separated from the carrier fluid channel1306 and the inspection circuit 1326 by a short neck region. Thediameter of the neck region is about 0.200 mm. However, other suitabledimensions are possible. The channels are oriented such that the threechannels containing aqueous solutions come together and intersect thechannel containing the carrier fluid at a 90 degree angle. Downstream ofthe mixer circuit, the solutions flow into the inspection circuit 1326.

FIG. 14 shows a representative example of a cross-section through acrystal card similar to the embodiment illustrated in FIG. 9. Thecrystal card 1400 is comprised of three layers 1420, 1430 and 1440.Layer 1430 comprises the ports as shown in FIGS. 7-9. Layer 1430 isabout 0.4 mm thick. Layer 1440 comprises the microfluidic channelcircuit and is about 1.5 mm thick at the edge. Layer 1420 comprises thepeelable layer attached to the bottom surface of the crystal card 1400,and is about 0.14 mm thick.

FIGS. 15A-15V illustrate a method 5000 for crystallizing molecules usinga nanovolume microcapillary crystallization system. From a start block,the method 5000 proceeds to a set of method steps 5002, defined betweena continuation terminal (“Terminal A”) and an exit terminal (“TerminalB”). The set of method steps 5002 describes the preparation of a crystalcard and the connection of the crystal card to a pump.

From Terminal A (FIG. 15B), the method 5000 proceeds to a set of methodsteps 5008 where the crystal card is manufactured from a suitablematerial, such as polydimethylsiloxane (PDMS) or plastic by injectionmolding. The method then returns to a point of invocation. The method5000 next proceeds to a set of method steps 5010 defined by acontinuation terminal (“Terminal A2”). The set of method steps 5010treats the microcapillary surface of the crystal card to reduce thesurface energy.

From Terminal A2 (FIG. 15C), the method 5000 proceeds to decision block5014 where a test is performed to determine whether the crystal card ismanufactured from plastic. If the answer to the test is NO, the methodproceeds to another continuation terminal (“Terminal A4”). If the answerto the test at decision block 5014 is YES, the method proceeds toanother decision block 5016 where another test is performed to determinewhether the plastic is polycarbonate. If the answer to the test atdecision block 5016 is NO, the method 5000 proceeds to anothercontinuation terminal (“Terminal A5”). If the answer to the test atdecision block 5016 is YES, the method 5000 proceeds to anothercontinuation terminal (“Terminal A6”).

From Terminal A4 (FIG. 15D), the method 5000 proceeds to block 5018where the method treats the crystal card as manufactured from PDMS. Themethod proceeds to block 5020 where the microcapillary surface istreated with a perfluorinated silane solution for 2 hours at roomtemperature. The method then proceeds to block 5022 where theperfluorinated silane solution is removed by vacuum. At block 5024, themicrocapillary surface of the crystal card is dried using a gas such asair under pressure at 5-10 psi for 1 hour. The method then returns tothe point from which the steps of Terminal A2 were invoked, and proceedsto another continuation terminal (“Terminal A3”). See block 5012.

From Terminal A5 (FIG. 15E), the method 5000 proceeds to block 5026where the method treats the crystal card as made of a plastic comprisingcyclic olefin copolymer (COC) or comparable plastic. At block 5028, themicrocapillary surface is treated with a reagent to reduce the surfaceenergy (hydrophobicity) of the plastic for 2 hours at room temperature.Suitable reagents for reducing the surface energy include fluorinatedcopolymer solutions, but other reagents are possible. Suitablefluorinated copolymer solutions include a two percent fluorinatedcopolymer solution in a fluorosolvent, such as Cytonix PFC 502AFA(Cytonix Corp., Beltsville, Md.). Cytonix PFC 502AFA is manufactured toadhere to polycarbonate and reduce the surface energy to 6-10 dyne/cm.To apply the fluorinated copolymer solution, the crystal card is filledfrom the outlet with the Cytonix PFC 502AFA solution. At block 5030, thefluorinated copolymer solution is removed by vacuum. At block 5032, themicrocapillary surface is dried using a gas such as air under pressureof 5-10 psi for 1 hour. The method 5000 then proceeds to block 5034where the crystal card is heated to 60° C. for 1 hour. The method thenreturns to the point of invocation of the steps of Terminal A2. Seeblock 5012 at Terminal A3.

From Terminal A6 (FIG. 15F), the method 5000 proceeds to block 5036where the crystal card is pre-chilled on ice. At block 5038, themicrocapillary surface is treated with a fluorinated copolymer solutionsuch as Cytonix PFC 502AFA for 2 hours on ice. The polycarbonate crystalcard inlets may be prone to cracking if incubated with the 502AFAsolution at higher temperatures. The method then proceeds tocontinuation terminal A5 where it skips to block 5030 and performs thesteps in blocks 5030, 5032, and 5034. The method then returns to a pointat which the steps of Terminal A2 were invoked. See Terminal A3 at block5012. The set of method steps at block 5012 couples the crystal card tothe pump.

From Terminal A3 (FIG. 15G), the method 5000 proceeds to block 5040where syringe 1 is filled with a buffer or aqueous solution. At block5042, syringe 2 is filled with a precipitant solution. At block 5044,syringe 3 is filled with a carrier fluid. A representative example of asuitable carrier fluid includes a fluorinated carbon solution. Suitableexamples of a fluorocarbon fluid include FC-40. FC-40 has a high surfacetension with the detergents used in solubilizing membrane proteins. Thesurface tension enables nanoplug formation and crystallization. In arepresentative embodiment, the carrier fluid is a fluorinated oil whichis immiscible with aqueous fluids. The carrier fluid surrounds andseparates the aqueous nanoplugs as they are formed, moving them forwardthrough the crystal card during the method. At block 5046, syringe 4 isfilled with a protein solution containing the protein of interest in asuitable buffer. At block 5048, suitable tubing such as Teflon® tubingis attached to the needle of each syringe. At block 5050, syringes 1 and2 are attached to pump 1, and syringes 3 and 4 are attached to pump 2.At block 5052, the tubing is connected to the crystal card via amacro-micro interface. Suitable connections for the macro-microinterface are described above. The method then proceeds to exit TerminalB.

From Terminal B, the method 5000 proceeds to a set of method steps 5004,defined between a continuation terminal (“Terminal C”) and an exitterminal (“Terminal D”). The set of method steps 5004 receivesinstructions to regulate fluid flow through the crystal card to obtaincrystals. From Terminal C (FIG. 15H), the method 5000 proceeds to a setof method steps 5054, defined by a continuation terminal (“TerminalC1”). The set of method steps 5054 configures the pump.

From Terminal C1 (FIG. 15I), the method 5000 proceeds to block 5060where the method receives instructions on the type of syringe pump modelto be controlled by the system. Suitable pumps include Harvard ApparatusTwin Syringe Pump Model 33 (Harvard Apparatus, Holliston, Mass.), whichhas been modified by the manufacturer to provide better accuracy. Asillustrated by FIG. 2, each syringe pump controls two syringes. At block5062, the method receives instructions on the serial communication portof a computer used to control the pump system. The communication portsare configured such that each syringe pump receives instructions at thesame time, thereby preventing time delays and allowing the solutions toflow through the crystal card simultaneously. The method proceeds toblock 5064 where the method receives instructions on the volume of eachsyringe connected to the pumps. At block 5066, the method determines thediameter of each syringe connected to the pumps. The method thenproceeds to return to a point at which the steps of the Terminal C1 wereinvoked.

From block 5054, the method 5000 proceeds to a set of method steps 5056defined by a continuation terminal (“Terminal C2”). The set of methodsteps primes fluids to the mixer circuit of the crystal card. FromTerminal C2 (FIG. 15J), the method 5000 proceeds to block 5068 where themethod receives instructions on which syringe will be used to dispensefluids into the mixer of the crystal card. At block 5070, the methodreceives instructions on the flow rate from each syringe. At block 5072,the method receives instructions on the volume of fluid to be dispensedby the syringe. At block 5074, the method dispenses or aspirates fluidfrom a fluidic channel upstream of the mixer circuit. The method thencontinues to another continuation terminal (“Terminal C4”).

From Terminal C4 (FIG. 15K), the method 5000 proceeds to decision block5076 where a test is performed to determine whether the syringe isdispensing an aqueous fluid. If the answer to the test at decision block5076 is NO, the method proceeds to another continuation terminal(“Terminal C5). If the answer to the test at decision block 5076 is YES,the method proceeds to block 5078 where the method receives instructionsto stop the aqueous fluid at the mixer circuit and before the fluidenters the inspection circuit. The method then continues to Terminal C2and repeats the above identified process steps for the next syringe.From Terminal C5 (FIG. 15K), the method 5000 proceeds to block 5080where the method receives instructions to stop the carrier fluiddownstream of the mixer circuit and slightly inside the inspectioncircuit. The method then proceeds to return to a point from which thesteps of Terminal C2 were invoked.

Digressing, an illustrative process for priming aqueous solutions andthe carrier fluid to the mixer of the crystal card will now be describedin detail. First, the empty crystal card mixer circuit is positioned onthe microscope stage for observation during priming. The method receivesinstructions to dispense a solution, for example buffer, from syringe 1to the mixer. The buffer is dispensed into the fluid channel connectedto syringe 1 until the user observes that the solution has reached theregion of the mixer just upstream of the junction between the fluidicchannels. The method then receives instructions to stop dispensing thesolution. Solution may be removed from the channel by instructing themethod to aspirate the reagent. It is suitable to refrain aqueoussolutions from entering the inspection circuit of the crystal card. Themethod is repeated for each of the three fluid channels connected tosyringes dispensing aqueous solutions; for example, syringe 4 (proteinsolution) and syringe 2 (precipitant solution). The carrier fluid isthen dispensed into the fourth fluid channel connected to syringe 3. Thecarrier fluid is dispensed into the fourth fluid channel until the fluidtravels through the mixer junction and just slightly enters theinspection circuit (fifth channel) of the crystal card. The method thenreceives instructions to stop dispensing the carrier fluid.

Returning to block 5056, the method 5000 proceeds to a set of methodsteps 5058 defined by a continuation terminal (“Terminal C3”). The setof method steps receives instructions to produce aqueous nanoplugs inthe inspection circuit of the crystal card. From Terminal C3 (FIG. 15L),the method 5000 receives instructions on which nanoplug formationprotocol will be performed at block 5082. The method then proceeds todecision block 5084 where a test is performed to determine whether theinstruction received was to perform the constant mode. If the answer tothe test at block 5084 is NO, the method proceeds to anothercontinuation terminal (“Terminal C6”). If the answer to the test atdecision block 5084 is YES, the method proceeds to block 5086 where themethod receives instructions on the flow rate for each syringe. Themethod then proceeds to block 5088 where the method receivesinstructions on the total volume of fluid to pass through the mixercircuit. At block 5090, the method produces aqueous nanoplugs inside theinspection circuit of the crystal card wherein each nanoplug is suitablyof equal size and has the similar concentration of protein andprecipitant. The method then proceeds to return to a point ofinvocation. From block 5058, the method proceeds to exit terminal D.

From Terminal C6 (FIG. 15M), the method 5000 proceeds to decision block5092, where a test is performed to determine whether the method wasinstructed to perform gradient mode. If the answer to the test in block5092 is NO, the method proceeds to another continuation terminal(“Terminal C7”). If the answer to the test in decision block 5092 isYES, the method proceeds to block 5094 where the method receivesinstructions on the maximum flow rate for the syringes with variableflow. In one embodiment, the variable flow syringes contain the bufferand precipitant. In another embodiment, syringes 1 and 2 are thevariable flow syringes. However, the method can designate any syringe tobe a variable flow syringe. In one embodiment, the combined flow rate ofthe variable flow syringes equals the maximum flow rate. For example, inone embodiment, the method provides instructions for the flow rate ofsyringe 1 to equal 2 μl/min, whereas the method provides instructionsfor the flow rate of syringe 2 to equal 0 (zero) μl/min. In thisembodiment, the maximum flow rate equals 2 ul/min (2+0 μl/min). Themethod then proceeds to block 5096 where the method receivesinstructions on the constant flow rate for the syringe controlling thecarrier fluid. In one embodiment, syringe 3 controls the carrier fluid.In one embodiment, the carrier fluid flow rate equals the total flowrate of the aqueous solutions (buffer, precipitant, and proteinsolutions). In another embodiment, the flow rate for the carrier fluidmay be selected to be slower or faster than the total flow rate of theaqueous fluids. Slower carrier fluid rates generate larger aqueousnanoplugs with smaller segments comprising carrier fluid betweennanoplugs. Faster carrier fluid rates generate smaller aqueous nanoplugswith larger carrier fluid segments between the nanoplugs. The methodthen proceeds to block 5098 where the method receives instructions onthe constant flow rate for the syringe controlling the protein solution.In one embodiment, syringe 4 controls the carrier fluid. In oneembodiment, the protein flow rate equals the sum of the flow rate of theother aqueous solutions (buffer and precipitant). Changing the flow rateof the protein solution changes the ratio of protein-to-crystallizationconditions in each nanoplug. The method then proceeds to block 6000where the method receives instructions on the total aqueous volume to bedispensed during a single iteration or cycle of the method. The methodthen proceeds to another continuation terminal (“Terminal C8”).

From Terminal C8 (FIG. 15N), the method 5000 proceeds to block 6002where the method receives instructions on the volume of each aqueousnanoplug that will be dispensed into the inspection circuit. At block6004, the method receives instructions on the total number of iterationsor cycles to be performed (i.e., the number of times the gradientscreening steps are repeated). In one embodiment, if the method receivesinstructions to run zero iterations, the pumps will stop when the totalaqueous volume selected at block 6000 is dispensed. In anotherembodiment, if the method receives instructions to run one or moreiterations, the pumps will stop when the process steps described abovehave been repeated the desired number of times. At block 6006, themethod reciprocally varies the flow rate of the buffer and precipitantsolutions such that the sum of the buffer and precipitant solution flowrates equals the maximum flow rate selected at block 5094. For example,in one embodiment, at block 5094 the method provides instructions forthe flow rate of syringe 1 to equal 2 μl/min and provides instructionsfor the flow rate of syringe 2 to equal 0 μl/min, such that the maximumflow rate equals 2 μl/min. When the method starts, the flow rate fromsyringe 1 will begin at 2 μl/min and ramp down to 0 μl/min, while theflow rate from syringe 2 will simultaneously ramp up from 0 μl/min to 2μl/min. At block 6008, the method produces a series of aqueous nanoplugsinside the inspection circuit wherein each drop is of equal size butvaries in the concentrations of protein and precipitant in each drop. Atblock 6010, the method terminates after the desired number of iterationsor cycles has been performed. The method then returns to block 5058where the method proceeds to exit terminal D.

From Terminal C7 (FIG. 15O), the method 5000 proceeds to decision block6012 where a test is performed to determine whether the method wasinstructed to perform hybrid mode. If the answer to the test at block6012 is NO, the method proceeds to another continuation terminal(“Terminal C9”). If the answer to the test at block 6012 is YES, themethod proceeds to another decision block 6014 where a test is performedto determine whether a precipitant cartridge has been prepared. If theanswer to the test at decision block 6014 is NO, the method proceeds toanother continuation terminal (“Terminal C10”). If the answer to thetest at block 6014 is YES, the method proceeds to another continuationterminal (“Terminal C11”).

From Terminal C9 (FIG. 15P), the method 5000 proceeds to decision block6016 where a test is performed to determine whether the method wasinstructed to perform the pulsatile mode. If the answer to the test atdecision block 6016 is NO, the method returns to Terminal C3 where theabove identified steps are repeated. If the answer to the test atdecision block 6016 is YES, the method proceeds to block 6018 where themethod receives instructions on performing the pulsatile mode. Themethod then returns to block 5058. From block 5058, the method exits toTerminal D.

From Terminal C10 (FIG. 15Q), the method 5000 proceeds to block 6020where a syringe is connected to tubing, such as Teflon® tubing,containing carrier fluid. The method then proceeds to block 6022 wherethe syringe is connected to a syringe pump. At block 6024, the methodreceives instructions to enter a defined volume, for example, about 40nL, and aspirates an air bubble of about 40 nL into the tubing. At block6026, the method aspirates a defined volume, for example, about 120 nL,of a precipitant solution into the tubing. At block 6028, the methodrepeats the above two steps until a suitable number of precipitants areloaded into the tubing. For example, a suitable number of precipitantscan range from 1-24 or more. At block 6030, the method aspirates carrierfluid, about 1 μL, into the open tip of the tubing. At block 6032, thetubing is connected to the precipitant inlet of the crystal card. Themethod then proceeds to continuation Terminal C11.

At Terminal C11 (FIG. 15R), the method 5000 proceeds to block 6034 wherethe method receives instructions on the starting flow rate of the buffersolution (syringe 1). At block 6036, the method receives instructions onthe change in the flow rate (step size) of the buffer solution. The stepsize is the change in the rate of flow that will be applied at each rampup or down of the method. At block 6038, the method receivesinstructions on the starting flow rate of the precipitant cartridge(syringe 2). At block 6040, the method calculates the change in the flowrate (step size) of the precipitant solution. In one embodiment, thestep size for the buffer equals the step size for the precipitant. Atblock 6042, the method sums the buffer and precipitant flow rates todetermine the total flow rate. At block 6044, the method receivesinstructions on the starting flow rate for the carrier fluid (syringe3). At block 6046, the method receives instructions on the change in theflow rate (step size) of the carrier fluid. The method then proceeds toanother continuation terminal (“Terminal C12”).

From Terminal C12 (FIG. 15S), the method 5000 proceeds to block 6048where the method receives instructions on the constant flow rate of theprotein solution (syringe 4). The method then proceeds to block 6050where the method receives instructions on the number of ramp up steps(rate of flow changes) for each precipitant. At block 6052, the methodsets the number of ramp down steps to equal the number of ramp up stepsfor each iteration or cycle of the method. At block 6054, the methodreceives instructions on the number of iterations or cycles to beperformed. In one embodiment, one iteration or cycle corresponds to asingle precipitant loaded in the precipitant cartridge. At block 6056,the method receives instructions on the duration of each ramp step. Forexample, in one embodiment, the duration of each ramp step is 1.5seconds. At block 6058, the method reciprocally varies the buffer andprecipitant flow rates such that the sum equals the starting rates. Themethod then proceeds to another continuation terminal (“Terminal C13”).

From Terminal C13 (FIG. 15T), the method 5000 proceeds to block 6060where the method varies the flow rate of the carrier fluid. The methodthen proceeds to block 6062 where the method produces a series ofnanoplugs inside the inspection circuit wherein each drop has equalamounts of protein and varying amounts of precipitant and buffer. In oneembodiment, the method provides a varied amount of precipitant with aconstant amount of protein for each cycle. Table 1 illustrates oneembodiment of the method described above for the hybrid mode. The methodthen proceeds to Terminal D.

TABLE 1 Values specified by the method in hybrid mode. One Carrier Totalcycle Buffer Precipitant Fluid Protein Sum Duration time up Step(ul/min) (ul/min) (ul/min) (ul/min) Aqueous (sec) (sec) 0 0.2 0.6 2.20.6 1.4 1.5 Ramp 1 0.3 0.5 2.0 0.6 1.4 1.5 up 2 0.4 0.4 1.8 0.6 1.4 1.53 0.5 0.3 1.6 0.6 1.4 1.5 4 0.6 0.2 1.4 0.6 1.4 1.5 6.0 Ramp 1 0.5 0.31.6 0.6 1.4 1.5 down 2 0.4 0.4 1.8 0.6 1.4 1.5 3 0.3 0.5 2.0 0.6 1.4 1.54 0.2 0.6 2.2 0.6 1.4 1.5 6.0 Step 0.1 0.1 0.2 stable size

From Terminal D at block 5004, the method 5000 proceeds to a set ofmethod steps 5006, defined between a continuation terminal (“TerminalE”) and an exit terminal (“Terminal F”). The set of method steps 5006performs diffraction experiments on the crystals obtained from thecrystal card. From Terminal E (FIG. 15U), the method 5000 proceeds todecision block 6064 where a test is performed to determine whethercrystals were extracted from the inspection circuit of the crystal cardprior to diffraction. If the answer to the test at block 6064 is NO, themethod proceeds to another continuation terminal (“Terminal E1”). If theanswer to the test at block 6064 is YES, the method proceeds to block6066 where a peelable layer is removed from the bottom surface of thecrystal card. In one embodiment, the peelable layer is bonded to theplastic part of the crystal card that contains the microfluidicchannels. The bond is designed to be strong enough to prevent fluid fromleaking out of the microfluidic circuit but weak enough to be manuallypeeled off. In one embodiment, the bond is a thermal bond. In anotherembodiment, the bond is a chemical bond. Removal of the peelable layerexposes the interior of the microfluidic channels of the crystal card,allowing access to the aqueous nanoplugs. In another embodiment, theaqueous nanoplugs that contain crystals are retained in the microfluidicchannels of the crystal card after the peelable layer is removed. Atblock 6068, the crystal formed in the inspection circuit is extractedfrom the crystal card using a cryoloop. In one embodiment, the cryoloopis a nylon cryoloop. At block 6070, the crystal is cryocooled, anddiffraction data is obtained. The method then proceeds to exit TerminalF where the method terminates execution.

From Terminal E1 (FIG. 15V), the method 5000 proceeds to block 6072where the crystal card containing crystals is mounted onto thegoniometer of an X-ray source. At block 6074, the method obtainsdiffraction data from crystals located in situ inside the inspectioncircuit. The method then proceeds to block 5006 and exit Terminal F. Themethod then terminates execution.

The above described crystal extraction steps can be used in combinationwith the gradient screening of various embodiments of the subject matterto generate crystals of methionine-R-sulfoxide reductase. Crystals wereremoved from the crystal card using a cryoloop and then cryocooled fordiffraction experiments. As an example, a 1.7 Å data set was collectedat SBC-CAT beamline 19BM located at the Advanced Photon Source atArgonne National Laboratories and the structure was subsequently solvedand refined. The final coordinates and structure factors were depositedto the Protein Data Bank (accession code 3CXK).

The crystal card of various embodiments of the subject matter is alsosuitable for in situ diffraction. In situ diffraction allows thecrystallographer to assess the quality of a crystal before being alteredby the cryoprotection process. For robust crystals, it can allowcomplete diffraction data to be collected. The crystal card issufficiently X-ray transparent to be mounted onto the goniometer of anX-ray source for diffraction data collection at room temperature. Forexample, a simple test was conducted to analyze the absorption of theX-Rays by the crystal card. The beam current in the ion chambernormalized to the APS ring current (I/I₀) was measured with and withoutthe crystal card inserted at a wavelength of 0.979261 A (12.66099 keV).I/I₀ without the crystal card measured 1.91671 E-6 and I/I₀ with thecrystal card measured 1.5511 E-6. This constitutes a 19% X-rayabsorbance by the crystal card. Further, the crystal card can betranslated along its X and Y axis to collect data from multiple crystalsto be combined for a complete data set. To demonstrate this technique, acrystal card containing Lysozyme crystals was mounted on the goniometerhead at NE-CAT beamline 24ID-C located at the Advanced Photon Source atArgonne National Laboratories. Data were collected at room temperaturefrom three crystals in the crystal card. Crystallographic data areprovided in Appendix A.

Regarding structure determination, data sets were collected at theAdvanced Photon Source: beamline 19BM at 100K for methionine-R-sulfoxidereductase and beamline 24-IDC at room temperature for lysozyme. Datawere integrated and scaled with HKL2000. For the lysozyme structure,intensities were integrated separately for each of the three data setsusing the mosflm package. The structures of lysozome andmethionine-R-sulfoxide reductase were solved by molecular replacementusing Molrep and PDB entries HEE and 3CEZ as the search models,respectively. Structures were refined with Refmac5 and model buildingwas performed with Coot.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

APPENDIX A Crystallographic Data: methionine-R- Lysozyme sulfoxidereductase Data Collection Unit Cell (Å) a = 79.18, a = 42.00, b = 45.17c = 38.38 c = 45.40, α = 88.4 β = 83.7, γ = 69.1 Space group P4₃2₁2 P1(No. 1) (No. 96) Resolution (Å) 50-1.90 50-1.70 Wavelength (Å) 0.979500.97932 Total Reflections 54,338 118,181 Unique Reflections 10,15132,539 I/(sigI)* 11.4 (2.9)  23.1 (2.2)  R_(merge) (%)* 13.7 (58.4)  6.8(42.3) Completeness (%)* 98.8 (98.5) 95.3 (87.4) Redundancy 5.4 (5.0)3.6 (3.3) Wilson B factor (Å²) 24.1 22.1 Refinement Resolution (Å)50-1.90 50-1.70 Reflections (working/test) 9,412/480   30,828/1,650 R_(working)/R_(free) (%) 19.6/23.0 16.6/19.9 Number of atoms(protein/water) 1001/45  2082/180  r.m.s. deviation bond length (Å)0.016 0.015 r.m.s. deviation bond angle 1.607 1.408 (degrees) Average Bfactor (Å²) (All atoms) 28.8 28.3 Average B factor (Å²) (Protein) 28.527.5 Average B factor (Å²) (Water) 35.9 37.0 Coordinate error (Å) Basedon R_(free) 0.149 0.095 Ramachandran Analysis (%) Most Favored (chainA/B) 89.4 91.7/90.8 Additionally Allowed (chain A/B) 10.6 7.3/8.3*Parenthesis indicates values for the 2.00 Å to 1.90 Å resolution shellfor lysozyme and 1.76 Å to 1.70 Å shell for methionine-R-sulfoxidereductase.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A proteincrystallization system, comprising: a pumping system; pieces of softwareconfigured to execute on the protein crystallization system to controlthe pumping system; and one or more crystal cards coupled to the pumpingsystem, each configured to house a mixer and a microfluidic capillarythat is coupled to the mixer to facilitate storage and inspection ofprotein crystallization.
 2. The protein crystallization system of claim1, wherein the pumping system includes a syringe pumping system or apressure pumping system, wherein the syringe pumping system includesfour-channel syringe pumps to regulate aqueous solutions being conveyedinto the one or more crystal cards through second, third, and fourthmicrofluidic channels, and fluorous solutions being conveyed into afifth microfluidic channel.
 3. The protein crystallization system ofclaim 1, wherein the pieces of software facilitate control of each pumpof the four-channel syringe pump and control of each channel of second,third, fourth, and fifth microfluidic channels to generate granulargradients of flow of aqueous solutions.
 4. The protein crystallizationsystem of claim 1, wherein the one or more crystal cards are formed frommaterials having properties that are selected from a group consisting ofX-ray transmissive, optical clarity, modable, chemical resistive,suitable surface energy, and a combination of two or more of theforegoing recited properties.
 5. The protein crystallization system ofclaim 1, wherein the mixer includes a junction of second, third, fourth,and fifth microfluidic channels where aqueous plugs are formed, thesecond, third, fourth, and fifth microfluidic channels being formed frommicrofluidic channels that are approximately 200 by 200 micrometers. 6.The protein crystallization system of claim 5, wherein the junctiondefines a hydrophobic surface that supports formation of aqueous plugs,which are approximately in a range of 10 nanoliters to 20 nanoliters,and wherein the microfluidic capillary transports the aqueous plugs awayfrom the junction.
 7. The protein crystallization system of claim 2,wherein the one or more crystal cards are formed from plastic configuredfor fine gradient screening and are alternatively formed fromPDMS/Teflon® configured for hybrid screening and membrane proteins. 8.The protein crystallization system of claim 1, further comprisingsyringes with needles coupled to the pumping system, and yet furthercomprising tubings having distal ends and proximal ends configured toact as macro-micro interface between the needles of the syringes and theone or more crystal cards, each tubing having an inner diameter of about360 micrometers and an outer diameter of about 760 micrometers, thedistal end of a tubing configured to slide onto a needle and theproximal end of the tubing configured to coupled to the one or morecrystal cards.
 9. A method for gradient screening, comprising:regulating aqueous streams by independently controlling each aqueousstream with a pumping system exercised by pieces of software; andmapping out crystallization phase space of a protein to illustratetransition from precipitation, to microcrystals, to single crystals in aprotein crystallization experiment.
 10. The method of claim 9, whereinthe act of regulating includes forming concentration gradients over aseries of aqueous plugs by changing flow rates of each aqueous stream.11. The method of claim 10, wherein the act of regulating includesregulating aqueous streams selected from proteins, crystallizationagents, fluorocarbons, precipitants, ligands, protein partners, DNAcomplexes, buffers, and cryoprotectants.
 12. The method of claim 11,wherein the act of regulating includes increasing a flow rate of anaqueous stream of a buffer when a flow rate of an aqueous stream of aprecipitant decreases so that a sum of flow rates remains constant. 13.A method for hybrid screening, comprising: pre-forming precipitantplugs; pre-forming plug spacers, each separating two precipitant plugsfrom each other; forming gradients by merging precipitant plugs, plugspacers, and a protein stream; mapping out crystallization phase spaceof a protein to illustrate transition from precipitation, tomicrocrystals, to single crystals in a protein crystallizationexperiment.
 14. The method of claim 13, wherein pre-forming plug spacersincludes pre-forming using gas bubbles.
 15. The method of claim 13,wherein forming gradients includes coordinating flow rate change betweena stream formed from the precipitant plugs, plug spacers, and a bufferstream.
 16. The method of claim 13, wherein each precipitant plug isabout 100 nanoliters.
 17. A method comprising: receiving a crystal cardwith capillaries; coating capillaries with a reagent to reduce a surfaceenergy; and removing the reagent.
 18. The method of claim 17, furthercomprising incubating the crystal card on ice for a predetermined numberof hours.
 19. The method of claim 17, wherein the capillaries includeinside surfaces and the act of coating capillaries includes coating theinside surfaces of the capillaries to reduce surface energy to about sixto ten dynes per centimeter.
 20. The method of claim 17, wherein thefluorinated copolymer solutions include two percent fluorinatedcopolymer solutions in fluorosolvent.
 21. The method of claim 17,wherein removing the fluorosolvent includes vacuuming the crystal card.22. The method of claim 17, further comprising an act of forcing clean,dry air through the crystal card, which is executed at five psi forabout one hour.
 23. The method of claim 17, further comprising an act ofbaking the crystal card, which is executed at about sixty degree Celsiusfor about one hour.
 24. The method of claim 17, further comprising:peeling a thin layer bonded to a substrate of a crystal card; extractingcrystals by a cryoloop from microfluidic circuitry housed on thesubstrate; cryocooling the crystals; and performing diffractionexperiments on the crystals to obtain diffraction data.
 25. The methodof claim 17, further comprising: mounting a crystal card withmicrofluidic circuitry to a goniometer; radiating the crystal card withX ray; and collecting diffraction data.
 26. The method of claim 25,further comprising translating the crystal card along x and y axes tocollect the diffraction data from multiple crystals stored by themicrofluidic circuitry.
 27. A crystal card, comprising: a substrateconfigured to house a mixer circuit and an inspection circuit; and alayer bonded to the substrate and configured to peel from the substrate.28. The crystal card of claim 27, wherein the layer is either thermallybonded to the substrate or chemically bonded to the substrate.
 29. Thecrystal card of claim 27, wherein the substrate and the layer are formedfrom a group consisting of an amorphous polymer, Cyclic OlefinCopolymer, a thermalplastic polymer, and Polycarbonate.
 30. The crystalcard of claim 27, wherein the substrate includes a thickness of aboutone millimeter and the layer includes a thickness in a range of about100 to 150 micrometers.
 31. The crystal card of claim 27, wherein themixer circuit includes first, second, third, and fourth summandchannels, each summand channel including a distal end and a proximalend, the distal end of each summand channel defining an openingconfigured to fluidly receive solutions, the proximal end of eachsummand channel defining an opening configured to fluidly communicateaqueous plugs or plug spacers, each summand channel having a first partbeing coupled to the distal end and a second part of the first, second,and third summand channels being coupled to the proximal end, the firstpart of each summand channel being spaced apart and oriented in parallelwith another summand channel, the second parts of the first and thirdsummand channels angled so that their proximal ends intersect, thesecond parts of the second and fourth summand channels continuing inparallel until the proximal end of the second summand channel intersectswith the proximal ends of the first and third summand channels to form avertex, a third part of the fourth summand channel continuing from thesecond part of the fourth summand channel at a ninety degree angle whereits proximal end intersects with the vertex at another ninety degreeangle.
 32. The crystal card of claim 31, wherein the first part of eachsummand channel is spaced apart from the first part of another summandchannel by about 4.50 millimeters.
 33. The crystal card of claim 31,wherein the substrate includes a first side, a second side, a thirdside, and a fourth side, the second side of the substrate being spacedapart from the distal end of the third summand channel by about 3.70millimeters, a length of the first and third side being approximately25.40 millimeters, a length of the second and fourth side being about76.20 millimeters, the first side of the substrate being spaced apartfrom the distal ends of the summand channels by about 6.00 millimeters.34. The crystal card of claim 33, wherein the second side of thesubstrate is spaced apart from the distal end of the third summandchannel by about 3.70 millimeters, the first side of the substrate beingspaced apart from the distal ends of the summand channels by about 6.00millimeters, the third side of the substrate being spaced apart from theinspection circuit by approximately 6.00 millimeters.
 35. The crystalcard of claim 31, wherein the inspection circuit includes a summationchannel, a serpentine body, and a tail channel which terminates in anopening configured to fluidly communicate, the summation channel beingcoupled to the vertex and continued in a direction that is collinearwith the proximal end of the fourth summand channel until the summandchannel reaches an axis that is collinear with the first part of thethird summand channel at which the summation channel makes a ninetydegree turn to join with the serpentine body of the inspection circuit.36. The crystal card of claim 35, wherein the serpentine body of theinspection circuit is formed from a compound curve having multipleconvex turnings coupled to each other by a serpentine channel tofacilitate fluid communication, one convex turning being spaced apartfrom a subsequent convex turning by about 53.31 millimeters, each convexturning having a length of about 2.00 millimeters.
 37. The crystal cardof claim 36, wherein a last convex turning of the serpentine body iscoupled to the tail channel.
 38. The crystal card of claim 37, wherein alength of the summation channel, the serpentine body, and the tailchannel is collectively about 67 centimeters, wherein cross-sectionaldimensions of the summation channel, the serpentine body, and the tailchannel are about 200 by 200 micrometers.
 39. The crystal card of claim27, wherein the substrate is configured to house two mixer circuits andtwo inspection circuits.
 40. The crystal card of claim 39, wherein thesubstrate houses multiple annular ports that project upwardly, some ofwhich multiple annular ports are adapted to fluidly receive solutions orfluidly communicate aqueous plugs or plug spacers.